Differential fumarate binding to Arabidopsis NADþ-malic enzymes 1 and -2 produces an opposite activity modulation

MARCOS A. TRONCONI; MARIEL GERRARD WHEELER; MARIA FABIANA DRINCOVICH; CARLOS SANTIAGO ANDREO

BIOCHIMIE

ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER

Lugar: Paris; Año: 2012 vol. 94 p. 1421 - 1430

0300-9084

Arabidopsis mitochondria contain two NADþ-malic enzymes, NAD-ME1 and NAD-ME2. These proteins have similar affinity for their substrates but display opposite regulation by fumarate, which strongly stimulates NAD-ME1 but inhibits NAD-ME2 activity. Here, the interaction of NAD-ME1 and -2 with fumarate was investigated by kinetic approaches, urea denaturation assays and intrinsic fluorescence quenching, in the absence and presence of NADþ. Fumarate inhibited NAD-ME2 at saturating, but not at low, levels of NADþ, and it behaved as competitive inhibitor with respect to L-malate. In contrast, NADME1 fumarate activation was higher at suboptimal NADþ concentrations. In the absence of cofactor, the fluorescence of both NAD-ME1 and -2 is quenched by fumarate. However, for NAD-ME2 the quenching arises from a collisional phenomenon, while in NAD-ME1 the fluorescence decay can be explained by a static process that involves fumarate binding to the protein. Furthermore, the residue Arg84 of NADME1is essential for fumarate binding, as the mutant protein R84A exhibits a collisional quenching bythis metabolite. Together, the results indicate that the differential fumarate regulation of ArabidopsisNAD-MEs, which is further modulated by NADþ availability, is related to the gaining of an allosteric site for fumarate in NAD-ME1 and an active site-associated inhibition by this C4-organic acid in NAD-ME2.